Three ionophores with intrinsic fluorescence signals will be studied by a combination of fluorescence-lifetime and stopped-flow rapid mixing techniques to gain information about their location within membranes and the rates and mechanisms of their transport reactions. The time-resolved fluorescence decay functions of the Ca2 ion ionophores X537A and A23187 and the K ion ionophore dansyl-lysine-valinomycin will be studied in phospholipid vesicle membranes and in organic solvents to establish the "effective polarity" of the environment in the membrane where the ionophore is bound and to determine if there is evidence for more than one environment suggesting that there is more than one location. The locations of the ionophores in phospholipid membranes will be determined by studying the time-resolved quenching and Forster-type quantum transfer reactions of the membrane-bound ionophores in the presence of quenchers, and donor and acceptor probes which are localized in various regions (aqueous phase, membrane surface and hydrocarbon chain region). Procedures based on comparison of the critical concentrations, Co, for transfer and self-transfer in solution and in membranes are described. These will be used as methods for determining localization of the donors, acceptors and (finally) the ionophores with respect to the membrane surface and the hydrocarbon chain region. Stopped-flow rapid kinetic methods are described for the direct measurement of the rate of crossing the membrane for the ionophores and their cation complexes. The rates of the trans-membrane step(s) for monovalent and divalent cations for the three ionophores. The role of lipophilic anions and the stoichiometry of the transported complexes will be investigated. The total fluorescence life-time and stopped-flow kinetic data will be combined to determine a detailed mechanism of action. The behavior of the ionophores in sarcoplasmic reticular membranes will be compared with that of phospholipid membranes.